Low back and neck pain are prevalent conditions in developed societies, and are linked to degeneration of the intervertebral disc (IVD), where aberrant cell biology, particularly in cells of the nucleus pulposus (NP) is thought to drive these degenerative changes. Recent microarray investigations have shed some light as to the phenotype of NP cells; an area of IVD research previously undefined, but the unique gene markers identified have yet to be investigated in a cohort of specimens representative of a range of ages and stages of degeneration, as well as localised to cells of the disc. Additionally, evidence regarding the expression of novel marker genes at the protein level is to date, largely unaddressed, but is imperative in order to fully characterise the adult human NP cell phenotype, as it is not understood whether all NP cells will express these proteins, or just a subset of cells. Similarly, few studies have assessed variations in the novel gene and protein expression profile of adult NP cells with ageing and degeneration, and it is therefore essential to determine this and identify whether the phenotype of NP cells alters as a consequence of such variables. Furthermore, the cervical NP cell phenotype is at present poorly characterised and it is therefore assumed that it is comparable to that of lumbar NP cells, with regards to chondrogenic and catabolic gene expression. Elucidation of whether NP cells derived from the two spinal regions are comparable with regards to phenotype may allow for the interchangeable use of cervical and lumbar IVD specimens for in vitro purposes. This study aimed to investigate the adult human NP cell phenotype, through comparison of gene expression levels determined by quantitative real-time PCR in specimens obtained from a range of adult human disc levels of varying ages and degenerative grades, and to assess the expression of novel NP and notochordal (NC) cell marker proteins by immunohistochemistry, in order to ascertain whether expression is ubiquitous, or whether discrete NP cell sub-populations exist in vivo, particularly with regards to NC cell marker expression using flow cytometry techniques. It has been postulated that the phenotype of NP cells is influenced by microenvironmental factors akin to those observed in vivo, and therefore an ex vivo model system for studying the effects of microenvironmental factors on the gene expression and histological profiles of cells within the NP was developed. Analysis of the cervical NP cell gene expression profile, and comparison to lumbar specimens confirmed that specimens from the two spinal regions were phenotypically similar and could therefore be used interchangeably. Following this, investigation of novel NP and NC cell marker expression in a cohort of degenerate human IVD samples identified differential expression of a range of markers, indicative of cell sub-populations existing within the tissue. It was subsequently demonstrated that discrete NC cell-like and NP cell-like cellular sub-populations exist, but crucially, provided support against the loss of NC cells from the NP with ageing, and intimate either that they differentiate into chondrocyte-like NP cells or that the subsets of NP cells are discrete with regards to ontogeny. The mechanisms underlying this required elucidation, and a novel ex vivo model system has been developed here, allowing for the assessment of NC cell phenotypic response to microenvironmental factors. Preliminary testing has indicated that hypoxic culture of porcine IVD explants results in alterations in the NP ECM and cellularity as compared to normoxic cultures, suggesting that microenvironment may influence alterations to NC cell phenotype. Taken together, these findings suggest that adult human NP tissues are comprised of heterogeneous cell populations, with differential expression of both NP and NC cell markers. Future work to investigate whether the functions of these phenotypically distinct cells differs may elucidate an optimal cell type for recapitulation in novel cell-based regenerative therapies for repair of the degenerate IVD.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:617962 |
| Date | January 2014 |
| Creators | Ludwinski, Francesca |
| Contributors | Hoyland, Judith; Richardson, Stephen; Gnanalingham, Kanna |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-cell-population-of-the-adult-human-nucleus-pulposus(a3e0ab47-25e2-43a5-aaf6-b0fb282d3e5b).html |
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